Tuberculosis remains the leading infectious cause of death worldwide, accounting for 2-3 million human deaths each year. Multi-drug resistant strains of M. tuberculosis have emerged that are significantly more difficult and expensive to treat. This program has focused on the development of genetic tools to analyze the molecular basis of drug resistance and virulence of M. tuberculosis. Relevant to both goals is the phenomenon of M. tuberculosis latency. Within the infected host, a subpopulation of tubercle bacilli enters a non-replicating latent state that confers phenotypic resistance to both antibiotics and host immune mechanisms. These latent "persisters" retain the ability to reactivate and cause progressive disease when host immunity is suppressed. In the last five years, the investigators have developed systems for complementation, allelic exchange, and transposon mutagenesis. These systems have allowed them to identify the, previously unknown, targets of the drugs isoniazid, ethionamide, and ethambutol. They have also established a molecular basis for the attenuation of an M. bovis strain as due to an amino acid substitution in a principal sigma factor of RNA polymerase and have developed auxotrophic vaccine strains of BCG. They plan to extend these studies by using phage-based systems for transposon mutagenesis and allelic exchange to generate libraries of signature- tagged mutants of M. tuberculosis with the goal of identifying genes required for virulence and persistence. In addition, they plan to use the transposon system to probe the cell surface of M. tuberculosis by characterizing phage-resistance mechanisms and studying the secretion pathways. Ultimately, the knowledge of mycobacterial virulence and persistence factors should lead to the development of novel drugs, vaccines, and immunotherapies to control tuberculosis.